First detection of emerging HoBi-like Pestivirus (BVD-3) among some persistently infected dairy cattle herds in Egypt.

Bovine viral diarrhoea virus (BVDV) is a serious veterinary health concern worldwide. We conducted this study to determine the prevalence of persistent infections (PI) and identify the current strain among some dairy cattle herds in Egypt. A total of 240 serum samples were collected from six Egyptian provinces. Between 2019 and 2020, samples were tested by Enzyme linked immunosorbent assay (ELISA) for detection of PI animals, and then molecular characterization was performed. Six calves were found PI with a prevalence of 2.5% (6/240). Using molecular characterization, HoBi-like Pestivirus (BVD-3) was successfully identified in Egypt for the first time. Based on the BVD-3 reference strains on Genbank, the detected strains had an identity ranging from 98.8 to 99.6%. Partial nucleotide sequence of the 5'UTR gene for six tested samples was submitted to Genbank with accessions: OM324396, OM324397, OM324398, OM324399, OM3243100, and OM3243101.

Increased genetic diversity of BVDV strains circulating in Eastern Anatolia, Turkey: first detection of BVDV-3 in Turkey.

Bovine viral diarrhea virus (BVDV) is a pathogen associated with loss of meat, milk, and reproductive performance in cattle across the world. There have been two types of BVDV identified worldwide: BVDV-1 and BVDV-2. However, a new type of BVDV, named HoBi-like pestivirus (BVDV-3), has been identified recently. BVDV presence in Turkey has been reported since the 1990s, but a mandatory vaccination program has not been implemented in Turkey so far. In serological studies conducted in Turkey for BVDV, reported seropositivity has been 50% on average. The aim of this study is to determine the genetic diversity of BVDV in blood and abortion materials from bovine in eastern Turkey. The presence of the virus was determined by antigen ELISA test. As a result of the phylogenetic analysis of 5'UTR, Npro and E2 genomic regions of the BVDV (n = 28), BVDV-1 (n = 25) was identified as the dominant type. In addition, BVDV-2 (n = 2) and BVDV-3 (n = 1) were determined which is the first report of HoBi-like pestivirus in Turkey. Although BVDV-1l (n = 19) was detected as the predominant sub-type of BVDV-1, 1a (n = 2), 1b (n = 1), 1c (n = 1), and 1d (n = 2) were also identified. In 2 samples, the BVDV-2 type detected was the 2a sub-type. In this study, it is emphasized that BVDV can be present in the abort materials as an agent and that it should be examined in the herd screening. In addition, it is understood that molecular epidemiological studies should continue for determining the genetic diversity of the viruses and that such studies should be carried out on the country basis. Necessary diagnostic programs should be developed for animals, which are imported or buying from other barns, and protection and control measures should be taken. The increase of reports on BVDV heterogeneity in Turkey and worldwide gets up related to the occurrence and spread of new BVDV types or variants, with potential implications for animal health and disease control.

Pestivirus infection in cattle dairy farms: E2 glycoprotein ELISA reveals the presence of bovine viral diarrhea virus type 2 in northwestern Italy.

BACKGROUND: Bovine viral diarrhea virus (BVDV) types 1 and 2 are members of the Pestivirus genus of the Flaviviridae family. This genus also includes the HoBi-like virus, tentatively classified as BVDV type 3. BVDV-1 is widely distributed in Italy despite the extensive use of BVDV-1-based vaccines, while BVDV-2 and HoBi-like Pestivirus have been detected occasionally. Monitoring the occurrence of sporadic or atypical pestiviruses is a useful approach to evaluate the need for additional vaccine strains that can be used in BVDV control programs. RESULTS: In this study we developed a multiwell antibody ELISA based on the recombinant E2 protein of the three bovine pestiviruses. We evaluated the assay's applicability for surveillance purposes using pooled milk samples, each prepared from a maximum of 35 lactating cows and collected from 176 dairy herds. As expected, the majority of the pooled samples reacted to a greater extent against the BVDV-1 E2 antigen. All three milk pools from a single farm reacted to the BVDV-2 antigen, however. Further analysis using spot tests, antigen detection, and sequence analysis of the 5'-UTR region confirmed the presence of five persistently infected calves carrying a BVDV-2a strain. CONCLUSIONS: This study highlights for the first time that sporadic circulation of BVDV-2 can be predicted by immunoenzymatic methods in the absence of specific vaccination.

Detection of emerging HoBi-like Pestivirus (BVD-3) during an epidemiological investigation of bovine viral diarrhea virus in Xinjiang: a first-of-its-kind report.

Xinjiang pastoral area is the second largest pastoral area in China, accounting for 26.8% of the available grassland area in the country, and the geographical advantage of cattle breeding industry is very obvious. Bovine viral diarrhea virus (BVDV) has always been one of the important viral diseases that have plagued the development of cattle farming industry in the world. As one of the main pastoral areas of China's cattle farming industry, the Xinjiang pastoral area has also been deeply affected. In this study, 6,153 bovine serum samples were collected from 18 large-scale cattle farms in 13 cities in Xinjiang. The antibodies and antigens of 6,153 and 588 serum samples were detected by serological detection methods, respectively. Ten serum samples, which were antigen-positive by ELISA, were randomly selected for RT-PCR detection, sequencing, and phylogenetic analysis of suspected HoBi-like Pestivirus (HoBiPeV) strains. The results showed that the positive rates of BVDV antibodies and antigens were 53.68% (3,303/6,153) and 6.12% (36/588), respectively. One of the 10 randomly selected seropositive samples was infected with the HoBiPeV strain. HoBiPeV, also referred to as BVDV-3, is an emerging atypical Pestivirus that occurs in cattle and small ruminants, and its clinical signs are similar to those of BVDV infection. Based on the whole genome of the BVDV-3 reference strain (JS12/01) on the GenBank, the homology of the detected strain was 96.02%. The whole genome nucleotide sequence was submitted to the GenBank database, and the gene accession number was obtained: OP210314. The whole genome of isolate OP210314 was 12.239 nucleotides and contained a 5'-UTR of 340 nucleotides, a 3'-UTR of 199 nucleotides, and a large open reading frame (ORF) encoding a polyprotein consisting of 3,899 amino acids. In conclusion, the prevalence rate of BVDV infection in Xinjiang dairy cows is high, and the genetic diversity is increasing. This study successfully identified and isolated HoBiPeV in Xinjiang for the first time, posing a potential threat to the cattle industry in Xinjiang.

Abortion outbreak in pregnant goats and cows with coinfection of 'Candidatus Mycoplasma haemobos' and HoBi-like pestivirus.

New emerging diseases, including 'Candidatus Mycoplasma haemobos' ('Ca. M. haemobos') and HoBi-like pestivirus in central China has been a huge challenge for ruminant production. From July to September 2022, an abortion outbreak affected 14 goat farms and 11 cattle farms in this area. To investigate whether the two pathogens are associated with the disease, samples were collected: Group 1 included 55 goat blood samples with foetal tissue samples and ticks on the skin; Group 2 included nine healthy goat blood samples; Group 3 included 36 cow blood samples with foetal tissue samples and ticks on the skin; and Group 4 included seven healthy cow blood samples. Then, these samples were analysed by serology, PCR, sequence analysis, and identification. A total of 339 Rhipicephalus (Boophilus) microplus and 61 Haemaphysalis longicornis ticks were identified in Group 1 and Group 3. By molecular detection, 32 'Ca. M. haemobos'-positive amplicons, and 27 HoBi-like pestivirus-positive amplicons were amplified from goats in Group 1; meanwhile, 20 'Ca. M. haemobos'-positive amplicons, and 18 HoBi-like pestivirus-positive amplicons were amplified from foetuses. Statistical significance (P = 0.002) and association (OR=7.556) between the 'Ca. M. haemobos' PCR results of foetus and goat samples were observed, and statistical significance (P = 0.017) and association (OR=4.271) between the HoBi-like pestivirus PCR results of foetus and goat samples were observed. These significances and associations were also observed in Group 3. In addition, coinfections were detected in Group 1 and Group 3. 'Ca. M. haemobos' was detected in both tick species. Further serological results revealed that the frequency of HoBi-like pestivirus was 20.0 % (11/55) in Group 1 % and 30.6 % (11/36) in Group 3 in central China. No ticks, pathogens, or neutralizing antibodies were observed in Group 2 or Group 4. This is the first molecular evidence of 'Ca. M. haemobos' and HoBi-like pestivirus natural coinfections in goats and cows with abortion in China. HoBi-like pestivirus and 'Ca. M. haemobos' can be transferred from goats/cows to their foetuses.

Whole-Genome-Sequence-Based Evolutionary Analyses of HoBi-like Pestiviruses Reveal Insights into Their Origin and Evolutionary History.

HoBi-like pestivirus (HoBiPeV), classified under Pestivirus H species, is an emerging cattle pathogen of high economic impact. However, the origin and evolution of HoBiPeV are not very clear due to a lack of full genomic sequences from diverse clades. This study aimed to determine full-genome sequences of HoBiPeV strains of three novel clades (c, d and e) and perform full-genome-based genetic and evolutionary analyses. Bayesian phylogenetic analyses herein confirmed the existence and independent evolution of four main HoBiPeV clades (a, c, d and e) globally, with genetic divergence ranging from 13.0% to 18.2%. Our Bayesian molecular clock estimates revealed that HoBiPeV most likely originated in India, with a dated tMRCA of 1938 (1762-2000), evidencing a more recent origin of HoBiPeV. The evolution rate of HoBiPeV was estimated to be 2.133 × 10-3 subs/site/year at full-genome level but varied widely among individual genes. Selection pressure analyses identified most of the positively selected sites in E2. Additionally, 21.8% of the ORF codon sites were found under strong episodic diversifying selection, providing first evidence of negative selection in HoBiPeV evolution. No recombination event was evident for HoBiPeV-c, d and e strains. These findings provide new insights into HoBiPeV origin and evolutionary history for better understanding the epidemiology and host-pathogen interactions and stimulate vaccine research.

Molecular characterization of recent HoBi-like pestivirus isolates from cattle showing mucosal disease-like signs in India reveals emergence of a novel genetic lineage.

Natural infections with HoBi-like pestivirus (HoBiPeV) have been detected in cattle in South America, Europe and Asia with a range of clinical manifestations including fatal mucosal disease (MD). In India, although HoBiPeV in cattle has been reported, there is no information on currently circulating HoBiPeV strains and associated severe clinical disease. Between September 2018 and December 2019, suspected cases of bovine viral diarrhoea with severe disease in cattle were noticed in farmers' small holdings in Tamil Nadu State. To determine the extent of pestivirus infection, blood, serum, nasal or oral swab samples of 46 cattle from 18 villages were tested. Based on the real-time RT-PCR, antigen ELISA and nucleotide sequencing results, pestivirus was detected in nine cattle from eight villages in two districts and all pestiviruses were identified as HoBiPeV. All nine HoBiPeV-infected cattle displayed clinical signs resembling MD and HoBiPeV isolates (n = 9) obtained were characterized at genetic and antigenic level. Phylogenetic analyses based on 5'-untranslated regions (5'-UTR), Npro and combined 5'-UTR-Npro gene sequences revealed that eight HoBiPeV isolates clustered into a clade, distinct from all reported HoBiPeV clades (a-d), whereas one belonged to HoBiPeV-d clade, thus providing evidence of emergence of a novel HoBiPeV clade (e). This was also supported by HoBiPeV-e clade-specific amino acid substitutions in Npro and the antigenic reactivity patterns. The study demonstrates the existence and independent evolution of five HoBiPeV clades (four main clades) globally and surprisingly three exclusive to India. Also we confirm first HoBiPeV occurrence in southern India with predominant prevalence of HoBiPeV-e strains. Besides demonstrating increased HoBiPeV genetic diversity, here we show association of HoBiPeV with severe clinical disease involving fatalities highlighting impact of HoBiPeV on cattle health. The emergence of a novel HoBiPeV lineage provides new insights on global HoBiPeV epidemiology and genetic diversity reiterating the need for continuous monitoring of HoBiPeV in India.

Different impact of bovine complement regulatory protein 46 (CD46bov) as a cellular receptor for members of the species Pestivirus H and Pestivirus G.

The genus Pestivirus within the family Flaviviridae comprises highly relevant animal pathogens such as bovine viral diarrhoea virus 1 and 2 (BVDV-1 and -2) classified into the two species Pestivirus A and Pestivirus B, respectively. First described in 2004, HoBi-like pestiviruses (HoBiPeV) represent emerging bovine pathogens that belong to a separate species (Pestivirus H), but share many similarities with BVDV-1 and -2. Additionally, two giraffe pestivirus (GPeV) strains both originating from Kenya represent another distinct species (Pestivirus G), whose members replicate very efficiently in bovine cells. In this study, we investigated the role of bovine complement regulatory protein 46 (CD46bov), the receptor of BVDV-1 and -2, in the entry of HoBiPeV and GPeV. For this purpose, bovine CD46-knockout and CD46-rescue cell lines were generated by CRISPR/Cas9 technology and subsequent trans-complementation, respectively. Our results provide strong evidence that the impact of CD46bov differs between viruses belonging to Pestivirus H and viruses representing Pestivirus G: CD46bov revealed to be a major cellular entry factor for HoBiPeV strain HaVi-20. In contrast, GPeV strain PG-2 presented as largely independent of CD46bov, suggesting a different entry mechanism involving other molecular determinants which remain to be identified. In addition, we demonstrated that, similar to BVDV-1 and -2, virus isolates of both Pestivirus H and Pestivirus G are able to adapt to cell culture conditions by using heparan sulfate to enter the host cell. In conclusion, our findings show that different bovine pestiviruses use diverse mechanisms of host cell entry.

Genetic and evolutionary analysis of emerging HoBi-like pestivirus.

HoBi-like pestivirus, an emerging species within the Pestivirus genus, is an important pathogen associated with a variety of clinical manifestations of ruminants, especially cattle. HoBi-like pestiviruses were identified in several countries and from different hosts, and raised concerns with regard to their acute and persistent infections, which is implicated in economic losses for cattle farmers. However, the transmission path, codon usage bias, and host adaptation of the virus has not been studied. Hence, we performed the analysis the spatio-temporal transmission based on the available 5'-UTR sequences of HoBi-like pestivirus, and then conducted codon analysis of the complete coding sequence of the virus. The results show the virus appeared in 1952 (95% HPD: 1905-1985) and may have originated in India. In addition, Italy is the hub for the spread of the virus. Moreover, six potential recombination events and two complex recombination events were discovered. Analysis of codon usage patterns revealed that the effective number of codon (ENC) values with an average of 50.85, and the codon usage bias is greatly affected by natural selection, which is different from the previous BVDV-1, 2. Finally, codon adaptation index (CAI) analysis shows that pigs may be the potential origin species of the HoBi-like pestivirus. These findings will contribute to more effective control of the spread of the virus, extend the knowledge about the genetic and evolutionary features of HoBi-like viruses and provide some information for vaccine research.

Do modified live virus vaccines against bovine viral diarrhea induce fetal cross-protection against HoBi-like Pestivirus?

Bovine Pestivirus heterogeneity is a major challenge for vaccines against bovine viral diarrhea (BVD). In breeding herds, fetal protection is a high priority issue. To some degree, fetal infections in vaccinated heifers have been attributed to the antigenic diversity of bovine Pestiviruses. The purpose of this study was to assess fetal protection against a divergent bovine Pestivirus (Hobi-like Pestivirus, HoBiPeV) with a commercially available modified live vaccine (MLV) claiming fetal protection against BVDV 1 and BVDV 2 up to one year after the first inoculation. Five vaccinated and four unvaccinated heifers were challenged by intranasal inoculation with the HoBiPeV Italy-1/10-1 strain between 82 and 89 days after insemination, i.e. between 4 and 6 months after vaccination. At challenge, neutralizing antibody titers to HoBiPeV in vaccinated heifers were low or even undetectable. Of the four unvaccinated heifers, one control animal aborted (fetus not available) and the remaining three gave birth to HoBiPeV positive calves. Among the heifers of the vaccinated group, one aborted the fetus in the sixth month of pregnancy, which tested Pestivirus negative, while three others gave birth to healthy, HoBiPeV negative calves; the remaining heifer delivered one HoBiPeV positive calf. The results suggest that the BVDV vaccine might be able to elicit a partial fetal protection against HobiPeV, even in absence of a strong specific antibody response.

HoBi-like pestivirus infection leads to bovine death and severe respiratory disease in China.

HoBi-like pestivirus is an emerging atypical pestivirus in cattle and small ruminants, causing clinical signs similar to those observed in bovine viral diarrhoea virus infections. Natural infection of HoBi-like pestivirus has been reported in cattle herds and small ruminants in multiple countries in South America, Europe and Asia. However, HoBi-like pestiviruses were only identified from contaminated bovine serum and small ruminants in China. So far, no clinical cases induced by HoBi-like pestivirus infection were reported in Chinese cattle herds. Here, for the first time, we reported natural infection of HoBi-like pestivirus in a cattle herd in China. Sick cattle with severe respiratory and diarrhoea and high fatality rate were found in a beef cattle herd in Shandong province in November 2017. RT-PCR, viral isolation, sequencing and phylogenetic analysis showed that the primary causative agent was HoBi-like pestivirus. The isolated HoBi-like pestivirus strain, SDJN-China-2019, shared 94.1%-97.5% homology with the LV168-20_16RN strain from Brazil in nucleotide of 5'UTR, Npro and E2 while it shared only 88.5%-92.1% homology with Asian HoBi-like virus strain Th/04-Khonkaen. Multiple unique mutations of amino acid were observed in Npro and E2 proteins of SDJN-China-2019, which were different from that of other reference strains. In summary, this study provides the first evidence of HoBi-like pestivirus infection in Chinese cattle herds, raising potential threat to the cattle industry in China.

Epidemiology of Bovine Pestiviruses Circulating in Italy.

Pestiviruses are widespread and economically important pathogens of cattle and other animals. Pestivirus A (formerly known as Bovine viral diarrhea virus 1, BVDV-1), Pestivirus B (Bovine viral diarrhea virus 2, BVDV-2), and Pestivirus H (HoBi-like pestivirus, HoBiPeV) species are infecting primarily cattle. Like other RNA viruses, pestiviruses are characterized by a high degree of genetic variability. This high rate of variability is revealed by the existence of a number of viral subgenotypes within each species. In cattle, the highest number of pestivirus subgenotypes has been documented in European countries, particularly in Italy. The aim of this review is to report an up-to-date overview about the genetic diversity of pestiviruses in Italian cattle herds. All three bovine pestiviruses species have been identified in cattle population with variable frequency and geographical distribution. The genetic diversity of Italian pestiviral strains may have diagnostic and immunological implications, affecting the performance of diagnostic tools and the full cross-protection elicited by commercially available vaccines. Implementation and strengthening of coordinated approaches for bovine pestivirus control in Italy are recommended. Therefore, it would be extremely important to increase control and restriction measures to the trade of cattle and biological products of bovine origin, including those containing fetal bovine serum.

HoBi-Like Pestivirus and Reproductive Disorders.

HoBi-like pestivirus (HoBiPeV) is an emerging group of pestiviruses that has been detected in cattle and other ruminants in South America, Europe, and Asia. Analogous to other bovine pestiviruses, namely bovine viral diarrhea (BVDV) 1 and 2, HoBiPeV is able to cause a variety of clinical forms that range from asymptomatic infections to fatal disease, having a great impact on cattle productions and causing substantial economic losses, mainly as a consequence of the occurrence of reproductive failures. The manuscript aims to provide an updated review of the currently available literature about the impact of HoBiPeV infection on cattle reproduction. The reproductive disorders observed in cattle due to natural and experimental infections caused by this virus are reported along with the few available in-vitro studies involving the reproductive tract. HoBiPeV should be considered among the bovine pathogens that impact on reproduction, but there is a need for more specific and sensitive diagnostic methods, while the cross-protection elicited by commercially available BVDV vaccines should be better investigated.

Evaluation of currently available bovine viral diarrhoea virus (BVDV) and HoBi-like pestivirus (HoBiPeV) specific diagnostic tests in detection of highly divergent HoBiPeVs in cattle.

The emergence of novel and divergent HoBi-like pestivirus (HoBiPeV) strains in cattle in Asia recently has raised concerns with regard to their reliable and accurate diagnosis. Hence, the aim of this study was to evaluate currently available BVDV diagnostic tests and HoBiPeV-specific diagnostic tests in detection of genetically divergent strains of HoBiPeV. One strain each of HoBiPeV-c and d were subjected to two BVDV diagnostic RT-PCR tests, one HoBiPeV specific RT-PCR test, three BVDV diagnostic qRT-PCR tests, one HoBiPeV specific qRT-PCR test and two BVDV antigen capture ELISAs. Archived cattle sera (n = 41) from farms with reports of HoBiPeV natural infection were assessed for detection of HoBiPeV antibodies by VNT and two commercial BVD antibody ELISA kits. BVDV diagnostic qRT-PCR tests had better sensitivity than BVDV diagnostic RT-PCR tests, while majority of them except a commercial kit showed a lower sensitivity for HoBiPeV-d strain. The HoBiPeV specific qRT-PCR test was found more sensitive than HoBiPeV specific RT-PCR but both had lower sensitivity for HoBiPeV-d strain, as displayed by primer/probe sequence mismatches. The BVDV Erns antigen ELISA detected both the strains of HoBiPeV, but with a lower sensitivity for HoBiPeV-d strain, whereas BVDV NS3 antigen ELISA failed to detect them even at a high HoBiPeV titre. Compared to VNT, commercial BVDV antibody ELISA showed low to moderate sensitivity in detection of HoBiPeV antibodies, with a failure rate of 31.25% for the whole virus antigen based ELISA and a failure rate of 56.25% for NS3 antibody ELISA. The present study demonstrated new challenges in HoBiPeV diagnosis indicating a need in improvement of both HoBiPeV specific diagnostic RT-PCR and qRT-PCR for better utility in HoBiPeV epidemiology and biological product safety. Although more studies are required, this study reinforces that combined use of BVDV Erns and NS3 antigen ELISA may have some utility in preliminary differentiation between HoBiPeV and BVDV infection in PI cattle. Additionally, we show that the comparative VNT has a better sensitivity in detection of HoBiPeV exposure and there is a need of robust antibody ELISA for reliable detection of antibodies against this emerging bovine pestivirus.

Serological survey for antibodies against pestiviruses in Wyoming domestic sheep.

Pestiviruses including Bovine viral diarrhea virus type 1 (BVDV-1), BVDV-2 and Border disease virus (BDV) have been reported in both sheep and cattle populations, together with the HoBi-like, an emerging group of pestiviruses. Pestivirus control programs in the United States have focused on the control of BVDV-1 and 2. The incidence of pestivirus infection in sheep in the United States and the risk of transmission between cattle and sheep populations are unknown. The aim of this study was to perform serological surveillance for pestivirus exposure in sheep from an important sheep producing state in the Unites States, Wyoming. For this, sera from 500 sheep, collected across the state of Wyoming (US) in 2015-2016, were examined by comparative virus neutralization assay against four species/proposed species of pestiviruses: BVDV-1, BVDV-2, BDV and HoBi-like virus. Rates of exposure varied between geographic regions within the state. The overall pestivirus prevalence of antibodies was 5.6%. Antibodies were most frequently detected against BVDV-1 (4%), and the highest antibody titers were also against BVDV-1. Data from this study highlights understanding of the dynamics of sheep pestivirus exposure, consideration of reference strains used for VN assays, transmission patterns, and potential vaccination history should be taken into account in implementation of control measures against pestiviruses in sheep and for successful BVDV control programs in cattle.

Genetic Diversity of Brazilian Bovine Pestiviruses Detected Between 1995 and 2014.

Pestivirus infections in ruminants result in significant economic losses worldwide. The aetiological agents are three species from the genus Pestivirus, family Flaviviridae, including bovine viral diarrhoea virus type 1 (BVDV-1), BVDV-2, border disease virus (BDV), and an atypical pestivirus named HoBi-like pestivirus. In this study, eighty-nine pestivirus isolates that were collected in Brazil between 1995 and 2014 and that originated from either cattle, fetal bovine serum (FBS) or as cell culture contaminants were genotyped based on a comparison of gene sequences from their 5' untranslated regions (5'UTR), N-terminal autoprotease (Npro ) and envelope glycoprotein 2 (E2). Of these isolates, 53.9% of the sequences were genotyped as BVDV-1, 33.7% as BVDV-2 and 12.4% as HoBi-like pestivirus. The prevalence of subgenotypes within the species was as follows: BVDV-1a (35.9%), BVDV-2b (31.4%), BVDV-1b (10.1%), BVDV-1d (6.7%), BVDV-2c (2.2%) and BVDV-1e (1.1%). BVDV-2c and BVDV-1e were detected for the first time in Brazil. This study revealed extensive genetic diversity among Brazilian pestivirus isolates, and the combination of pestiviruses that was detected is unique to Brazil. This information may serve as a foundation for designing and evaluating diagnostic tools and in the development of more effective vaccines; therefore, it may potentially contribute to pestivirus control and eradication.

Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus.

HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples.

Clinical Presentation Resembling Mucosal Disease Associated with 'HoBi'-like Pestivirus in a Field Outbreak.

The genus Pestivirus of the family Flaviviridae consists of four recognized species: Bovine viral diarrhoea virus 1 (BVDV-1), Bovine viral diarrhoea virus 2 (BVDV-2), Classical swine fever virus (CSFV) and Border disease virus (BDV). Recently, atypical pestiviruses ('HoBi'-like pestiviruses) were identified in batches of contaminated foetal calf serum and in naturally infected cattle with and without clinical symptoms. Here, we describe the first report of a mucosal disease-like clinical presentation (MD) associated with a 'HoBi'-like pestivirus occurring in a cattle herd. The outbreak was investigated using immunohistochemistry, antibody detection, viral isolation and RT-PCR. The sequence and phylogenetic analysis of 5'NCR, N(pro) and E2 regions of the RT-PCR positive samples showed that four different 'HoBi'-like strains were circulating in the herd. The main clinical signs and lesions were observed in the respiratory and digestive systems, but skin lesions and corneal opacity were also observed. MD characteristic lesions and a pestivirus with cytopathic biotype were detected in one calf. The present study is the first report of a MD like presentation associated with natural infection with 'HoBi'-like pestivirus. This report describes the clinical signs and provides a pathologic framework of an outbreak associated with at least two different 'HoBi'-like strains. Based on these observations, it appears that these atypical pestiviruses are most likely underdiagnosed in Brazilian cattle.

HoBi-like pestivirus experimental infection in pregnant ewes: Reproductive disorders and generation of persistently infected lambs.

In order to evaluate sheep as experimental model to test the efficacy of HoBi-like pestivirus vaccines for cattle, 10 sheep at different stages of pregnancy (30 or 50 days) were experimentally infected with the Italian prototype isolate Italy-1/10-1. Irrespective of the stage of pregnancy, virus inoculation resulted in reproductive failures, consisting of abortion, stillbirths or birth of weak or persistently infected (PI) lambs. Aborted fetuses, stillborn and dead lambs displayed extensive histopathological changes, consisting of hemorrhages, congestion and mononuclear infiltration in major organs. Pestiviral antigens were detected by immunohistochemistry in most tissues with remarkable signals in lungs and kidneys. PI lambs were constantly viremic, shed the virus through the nasal secretions and feces and, in all cases but one, did not have detectable HoBi-like pestivirus antibodies before the assumption of colostrum. The single seropositive infected lamb showed low-titer viremia and viral shedding that ceased only several weeks after the 3-month observation period. The study proves that sheep are susceptible to the reproduction failures caused by HoBi-like pestivirus infection and can serve as a suitable model for the evaluation of the fetal protection induced by homologous experimental vaccines.